Abstract

Advances in synthetic biology are contributing to diverse research areas, from basic
biology to biomanufacturing and disease therapy. We discuss the theoretical foundation,
applications, and potential of this emerging field.

Review

Synthetic biology is an emerging field of interdisciplinary research that seeks to
transform our ability to probe, manipulate, and interface with living systems by combining
the knowledge and techniques of biology, chemistry, computer science, and engineering.
Its main aim is to increase the ease and efficiency with which biological systems
can be designed, constructed, and characterized. Core efforts in the field have focused
on the development of tools to support this goal, including new approaches to biological
design and fabrication. Although the first generation of synthetic systems demonstrated
genetic circuits that encode dynamic behavior, cellular computational operations,
and biological communication channels, more recent research has focused on implementing
synthetic biological devices and systems in diverse applications, including disease
therapy, environmental remediation, and biosynthesis of commodity chemicals. As the
field matures, synthetic biology is advancing biological frontiers by expanding biomanufacturing
capabilities, developing next-generation therapeutic approaches, and providing new
insights into natural biological systems. Here, we review the theoretical foundations,
diverse tool kits, and engineered systems that have emerged from synthetic biology
and discuss current as well as potential future applications, which include in-depth
studies of basic biology (such as understanding endogenous signaling pathways and
feedback circuits) and new frontiers in health and medicine (such as identification
of diseased cells and targeted therapeutics).

Conceptual frameworks for biological design

A central aim of synthetic biology is to increase the ease and efficiency with which
biological systems can be designed, constructed, and characterized. Although the manipulation
of biological organisms and molecular pathways long preceded the emergence of synthetic
biology, the engineering of biological systems has been a largely ad hoc exercise. A main reason is that biology is inherently diverse, mutable, and context
specific. Natural biological substrates, including genetic elements such as promoters
and genes, do not always behave predictably when implemented in different combinations,
and details such as how individual parts are physically connected can vary widely
across different construction methods. As a result, the components designed and assembled
for one biological system often cannot be predictably reused in another system. Synthetic
biology seeks to address this challenge by implementing a more 'engineering-ready'
conceptual framework that emphasizes the need to generate and report biological constructs
in a manner that is conducive to their understanding and utilization by a broad community
of researchers.

The application of engineering tools such as abstraction, decoupling, and standardization
was proposed early in the emergence of synthetic biology to support the efficiency
and scaling of the biological system design process [1]. An abstraction hierarchy that dissects the engineering process into several design
levels - DNA, parts, devices, and systems - provides synthetic biologists with a means
to manage complexity and distribute tasks. The design process at each abstraction
level can be performed relatively independently of the other levels, and detailed
information critical to one abstraction level need only be considered by designers
operating at that level. This division of labor reduces the amount of information
that each designer must be expert in to successfully design a part, device, or system.

Decoupling refers to the strategy of partitioning a complicated problem into simpler
tasks that can be tackled separately and assembled into a complete solution. The separation
of design and fabrication processes is an important example of decoupling supported
by advances in design tools and fabrication platforms. The increasing efficiency and
decreasing cost of DNA synthesis allow synthetic biologists to design novel systems
with the confidence that DNA components can be readily synthesized by commercial sources.
Furthermore, advances in DNA sequencing and synthesis provide researchers with access
to biological components encoding functions of interest using sequence information
deposited in databases, eliminating the need for physical exchange of genetic materials.

Standardization takes several forms, including standardization of physical assembly,
functional assembly, and characterization/measurements. Early physical assembly standards
used biological parts flanked by standardized sequences, enabling the interchangeable
combination and sequential assembly of parts conforming to the specified standard
through a constant restriction-enzyme/ligation-mediated cloning strategy [2,3]. Significantly less progress has been made in the field on functional assembly standards,
which focus on identifying sequence interfaces between two types of parts (for example,
ribosome binding site (RBS) and gene) that allow functional coupling and predictable
activity, independent of the specific sequence of each part. Several early physical-assembly
strategies encountered obstacles because the proposed standards impaired the functional
assembly of parts by requiring the insertion of standard sequences between each part.
In response, the field is shifting to assembly methods that do not require restriction-enzyme-mediated
cloning [4,5]. Finally, technical measurement and reporting standards have been proposed to eliminate
discrepancies that result from disparate experimental methods and to provide more
reliable and thorough characterization data [6]. Standardized characterization data will support reliable sharing and reuse of parts,
devices, and systems such that new designs can build on the foundation of previous
work and move beyond the ad hoc model of system development.

Advances in fabrication methods for genetic systems

As synthetic biological systems become increasingly sophisticated, fabrication methods
with larger capacities, greater precision, higher speed, and lower cost have become
increasingly important. Outpacing the development of novel parts and devices, a number
of groundbreaking fabrication techniques have been demonstrated in recent years, allowing
researchers to focus on system design while outsourcing or performing system fabrication
with higher efficiencies than was previously possible. Advances in multiplex oligonucleotide
synthesis and assembly with microfluidic arrays have allowed cheaper de novo synthesis of gene-length fragments [7-9]. Furthermore, several techniques have been developed for the assembly of large DNA
fragments, moving the field beyond laborious and time-consuming molecular cloning.

For example, transformation-associated recombination (TAR) in the yeast Saccharomyces cerevisiae has been used to construct yeast artificial chromosomes encoding genes and pathways
isolated from several different organisms [4,10]. Yeast artificial chromosomes can be further modified with bacterial artificial chromosome
sequences to transfer the constructs to bacteria and subsequently to mammalian cells
[11]. Enzymatic in vitro assembly methods, such as one-step isothermal DNA assembly, can allow DNA molecules
of several hundred kilobases to be assembled without restriction-enzyme-mediated digestion
[5,12]. A combination of in vitro and TAR-based assembly methods was used to assemble and clone the first bacterial
genome from chemically synthesized oligonucleotides [13]. However, large sets of parts encoding similar functions with distinct sequences
are needed to avoid undesired recombination events between components that share similar
sequences when assembling large genetic systems with recombination-based strategies.

In addition to DNA synthesis and assembly, methods have been developed for high-throughput
genome modification. Multiplex automated genome engineering (MAGE) uses the bacteriophage
λ-Red single-stranded-DNA-binding protein β to achieve allelic replacement in Escherichia coli. This process can greatly accelerate the optimization of biological systems and metabolic
pathways, provided that the target genes are known and that an efficient screening
method is in place to identify the desired variants within the diverse libraries generated
[14]. An alternative method termed trackable multiplex recombineering (TRMR) has been
developed to support applications in which a priori knowledge of which target gene to modify is lacking, enabling rapid mapping of genes
and quantification of population dynamics [15]. A complementary technology called hierarchical conjugative assembly genome engineering
(CAGE), which has been used to combine portions of a genome that have been modified
by MAGE, was also recently described [16]. Although genome modification has been reported in yeast [17], most high-throughput methods have been limited to demonstrations in E. coli and the extension of these technologies to mammalian cells remains an important challenge.

Constructing the toolbox: synthetic biological parts and devices

Over the past decade, the synthetic biology community has built a large collection
of biological parts and devices with diverse functions [18] (Table 1). One focus has been the development of control devices that alter protein levels
in response to prescribed input signals, supporting the ability to reprogram cellular
behavior. Most systems demonstrated so far use transcription-based control, incorporating
constitutive and inducible promoters from a small set of well-characterized parts,
such as the tetracycline- and isopropyl-β-D-thio-galactoside (IPTG)-inducible promoters. However, inadequacies in these often-used
promoters, including inducer toxicity and limited selection of input choices and response
profiles, have motivated the development of libraries of constitutive promoters with
varying strengths [19,20] and the generation of new inducible promoters responsive to small molecules [21], gaseous acetaldehyde [22], and light [23]. The expanding collection of promoter parts increases the range of stimuli and expression
levels that can be achieved, providing greater flexibility in controlling the behavior
of biological systems.

Table 1. The synthetic biology toolbox: common components used in synthetic biological systems

As the diversity of gene regulatory processes in natural biological systems comes
to light, efforts have also been directed to developing control devices that act through
posttranscriptional and posttranslational mechanisms. In addition to parts such as
degradation tags [24,25] and split inteins [26,27], non-coding regulatory RNAs have been used to construct a number of control devices
[28]. In one example, microbial gene expression was regulated by engineered RNA-responsive
regulators (termed 'riboregulators') that modulate translation initiation by either
obstructing or releasing the RBS of a target gene in response to the presence of a
separately transcribed RNA sequence [29]. Researchers have demonstrated the utility of riboregulators in a variety of applications,
including protein localization studies, perturbation of stress response networks,
and programmable cell killing [30]. RNA-based devices responsive to small-molecule and protein inputs have also been
demonstrated, exerting control over both transgenic and endogenous protein expression
in bacteria, yeast, and mammalian cells [31-33], leading to applications ranging from bacteria-mediated detection and breakdown of
pesticides [34] to disease-marker detection and cell-fate regulation in mammalian cells [35]. The unique properties of RNA-based control devices - including ease of design and
construction, small genetic footprint, high energy efficiency, fast regulatory time
scales, and the ability to tailor input responsiveness and regulatory stringency -
have made RNA a versatile substrate for designing programmable control systems.

In addition to controlling protein levels, synthetic biologists have developed tools
to modulate the spatial organization of protein molecules inside cells, resulting
in new strategies for regulating or rewiring cellular activities encoded in metabolic
and signaling pathways [36]. In one example, researchers constructed synthetic feedback loops within the yeast
mating mitogen-activated protein (MAP) kinase pathway by recruiting modulator proteins
to the pathway scaffold protein Ste5 through fusing leucine zipper domains to each
component, and demonstrated circuits with pulse generator, accelerator, delay, and
ultrasensitive switch functions [37,38]. In another example, synthetic protein scaffolds that spatially recruit metabolic
enzymes were implemented in E. coli, enabling the stoichiometric optimization of three mevalonate biosynthetic enzymes
and achieving a 77-fold increase in product titer while avoiding cellular toxicity
caused by the accumulation of a pathway intermediate [39]. As an alternative to protein-based scaffolds, rationally designed RNA strands have
recently been shown to assemble into higher-order structures, including sheets and
nanotubes, inside bacterial cells [40]. An RNA scaffold was applied to a two-enzyme hydrogen biosynthesis pathway and shown
to increase hydrogen production by up to 48-fold compared with an unscaffolded system
[40]. These examples highlight the utility of spatial engineering in enhancing and modifying
biological pathways.

Synthetic gene circuits

One of the hallmarks of synthetic biology has been the drive to engineer biological
systems from the bottom up. Model-driven design of synthetic gene circuits has demonstrated
the ability to build circuits of specified function [41-43]; differences between models and realized circuits have illuminated important and
unique aspects of biological system behavior, such as the effects of degradation processes,
cooperativity, and noise [44-46]. In addition to inspiring the design of more robustly operating systems, the insights
gained through synthetic approaches have contributed to our understanding of natural
biological systems [47].

Genetic circuits encoding dynamic behaviors

The construction of two genetic circuits encoding dynamic behavior, the repressilator
[44] and the toggle switch [48], marked two early efforts in genetic circuit design in synthetic biology. The repressilator,
a three-ring oscillator built from a loop of three repressor-promoter pairs, demonstrated
that an oscillatory response could be generated from biological parts not found in
natural biological oscillators. However, the synthetic oscillator showed fluctuations
and heterogeneity not observed in natural circadian oscillators, which generally use
interlinked positive and negative feedback loops [45]. Subsequent generations of oscillators have interlinked positive and negative transcriptional
feedback loops of different strengths to drive more robust oscillatory dynamics with
tunable periods and amplitudes in bacterial [42] and mammalian cells [49] (Figure 1a). In the post-genomics era, the importance of precise timing and coordination of
gene expression in regulating systems-level behavior is increasingly appreciated.
Well-defined, synthetic gene circuits that control the temporal profile of gene expression
can elucidate the contributions of expression dynamics to natural time-dependent processes,
such as cell signaling, cell-fate determination, and development. Synthetic gene circuits
also offer the potential to develop genetically encoded strategies for intervening
and controlling these natural processes.

Figure 1.Synthetic circuits that perform diverse functions can be coupled to achieve higher-order
responses. (a) Interlinked positive and negative feedback loops of different strengths drive an oscillatory
response. Arabinose-responsive transcriptional activator (AraC) expression positively
modulates gene expression and results in a positive feedback loop, whereas the isopropyl-β-D-thio-galactoside (IPTG)-responsive inhibitor of the lac promoter (LacI) inhibits expression and generates a negative feedback loop. The small-molecule
inducers arabinose and IPTG modulate the strength of these feedback loops [42]. GFP, green fluorescent protein. (b) A mammalian AND gate composed of RNA interference (RNAi) target sites evaluates small
interfering (si)RNA inputs. Unique RNAi target sites are placed in the 3' UTR of two
lacI genes, and LacI regulates the expression of a fluorescent reporter, resulting in an
AND logic evaluator for the siRNA inputs m1 and m2 [52]. YFP, yellow fluorescent protein. (c) Quorum-sensing circuitry allows population control. Cell density is broadcast by the
diffusible small molecule acyl-homoserine lactone (AHL), which is synthesized by the
enzyme LuxI (X). As cell density and AHL concentration increase, LuxR (R), a transcriptional
regulator, binds AHL and initiates expression of a 'killer' gene (encoding CcdB, a
lethal protein that targets the DNA gyrase complex), ultimately reducing the steady-state
cell density [58]. (d) Interlinking positive and negative feedback loops with communication circuitry enables
oscillation synchronization across a population of cells. Expression of R positively
regulates expression of X, R, GFP, and AiiA (A), an enzyme that degrades AHL. As A
increases in concentration, it degrades AHL and negatively modulates protein expression
levels [62]. (e) Combining logic processing with communication circuitry enables a synthetic biological
edge detection system. The expression of X and the transcriptional repressor cI (Y)
is turned ON in cells in the dark region, where Y represses the expression of the
pigment-producing protein (pigment: β-galactosidase, an enzyme that cleaves a substrate
to produce a black pigment). However, diffusion of AHL synthesized by cells in the
dark region activates R in cells at the edge of the light region (where Y is turned
OFF), thus turning ON expression of pigment only in cells along this edge [63].

Encoding cellular logic and computing functions

Genetic circuits that perform computations and logical evaluations of cellular information
provide the ability to assess intracellular states and environmental signals. They
transmit this information into changes in cellular function, such as production of
easily assayed readouts, activation of metabolic pathways, or initiation of cell-fate
decisions. Towards this goal, genetic circuits and devices capable of performing logical
evaluations have been built to detect small molecules (using tandem promoter systems
[50] and RNA devices [51]), and small RNAs such as small interfering (si)RNAs (using tandem RNA interference
(RNAi) target sites [52]) (Figure 1b). These various schemes have demonstrated the classic NOT, OR, NOR, and AND gates
that are used to build larger logic evaluators and computations.

Methods for counting and maintaining memory of system states will enable a broader
spectrum of intracellular computing. A genetic circuit that can count up to three
exposure events to a small-molecule inducer was built in bacteria by nesting polymerase-promoter
pairs controlled by riboregulators responsive to an inducible transactivator [53]. Although this system captured brief induction pulses, system performance was highly
dependent on pulse duration and frequency. The incorporation of genetic memory offers
an alternative strategy to increase the robustness of counting events over longer
time frames. A three-event counter circuit was demonstrated by using DNA recombinase-based
cascades that record each event as a permanent change to the DNA, where the output
of each recombinase event would 'prime' the next promoter-recombinase pair in the
circuit [53]. Synthetic networks of feedback loops have been built as memory circuits that lock
a system in one state through sustained production of proteins following a transient
signal that initiates the state. For example, toggle switches engineered to show bistability
in bacteria [48] and mammalian cells [54] use architectures of mutually inhibitory feedback loops to achieve reversible memory
of small-molecule pulses. As another example, a positive feedback loop built from
a synthetic transcriptional activator cascade demonstrated heritable memory over many
generations in yeast [55].

One recurrent limitation in adapting biological systems to perform computation through
the rules of binary logic is the analog nature of the responses. In particular, gene
expression leakage in the OFF state can contribute to improper input processing and
high basal output, diminishing an evaluator's signal-to-noise ratio [48,53,55]. In addition, control of highly lethal proteins and proteins that mediate irreversible
genetic changes requires stringent OFF states. To address this issue, researchers
have layered transcriptional and posttranscriptional control elements within genetic
circuits to provide strategies for achieving stringent regulation of transgenes in
mammalian [56,57] and bacterial cells [30]. In one example, an inducible promoter was layered with repressible expression of
a small hairpin (sh)RNA to achieve undetectable expression levels of the highly lethal
diphtheria toxin in the OFF state, thus enabling induced cell death only in the ON
state [56]. Although tight OFF states are desirable for binary computing, biological computing
necessarily exploits the analog and tunable nature of gene expression. Connecting
logical circuit outputs to changes in cellular state requires the ability to both
identify thresholds of expression at which cellular behavior diverges and tune the
output to cross that threshold when triggered. Combining the computational ability
of logical evaluators with improved strategies for leakage minimization and output
tuning should enable more robust computing. These tools can expand our ability to
detect and treat diseases by increasing diagnostic certainty and improving precision
in gene expression, and can also be used to probe previously inaccessible information
sets, such as the temporal and spatial profiles of particular developmental genes,
which will inform our fundamental understanding of biology.

Communication circuits supporting more complex behaviors

Communication systems are required to coordinate events and tasks between different
cells in a population. Synthetic communication circuits have been engineered in bacteria
using various bacterial quorum-sensing systems. In these systems, a lactone signal
is broadcast with increasing strength as cell density increases. At a given threshold
level, lactone binds and activates a transcriptional regulator, upregulating the expression
of a target gene. Broadcasting and receiving can be incorporated within a single cell
population or distributed between 'sender' and 'receiver' cells. Incorporating both
functions in a single population programmed to regulate a killer gene resulted in
population control and demonstrated how population heterogeneity can be exploited
to achieve a robust population response [58] (Figure 1c). Segregating tasks and localizing the sender population established a radial gradient
of signaling molecules. Coupling the quorum-sensing circuitry to a band-pass circuit,
which detects a specified range of input concentrations, achieved formation of various
radial patterns in the receiver cells [59]. In addition, connecting bacterial quorum systems to synthetic circuits has demonstrated
dual-population consensus response and symbiosis in biofilms [60,61] and synchronized genetic clocks [62] (Figure 1d). Finally, coupling a light-responsive device [23] to logic-processing circuitry and a communication module resulted in a biological
edge detector [63] (Figure 1e). These examples demonstrate how synthetic circuits can distribute and coordinate
computational tasks across a population of cells to achieve complex responses similar
to what is observed in natural pattern formation and development.

Beyond bacterial systems, mammalian receiver cells have been engineered to respond
to volatile chemical signals [64] and metabolic conditions [65] using engineered synthetic promoters. These receivers can potentially be paired with
various processing circuits and sender cells to generate synthetic hormone-signaling
systems and synthetic ecosystems. The eventual coupling of metabolic functions and
cell-fate circuitry to synthetic hormone-signaling systems will enable spatial patterning
of cell differentiation and timing of coordinated cellular responses, a requisite
for complex tissue formation and function.

Moving towards real-world applications

Despite remarkable advances in the design and construction of increasingly sophisticated
genetic circuits over the past decade, the transition of these systems to real-world
applications has been constrained by the limited availability of devices that can
connect synthetic circuitry with information in living systems. However, synthetic
biologists are developing new ways to connect natural and engineered systems. For
example, exploiting existing connections between synthetic circuitry and intracellular
information, researchers have used the natural correlation between DNA damage and
proteolysis of the ON state inhibitor λ cI in a genetic toggle switch to record transiently
induced DNA damage through the formation of a biofilm [66]. Taking another approach, researchers have constructed synthetic sensor devices from
natural components, such as promoter-repressor pairs [67,68], signaling pathway components [69], and small RNAs and their target sites [52,57], to extract information from biological systems. As the range of sensor devices,
processing circuitry, and output modules expands, synthetic biology is poised to address
a broad scope of biological, medical, and biotechnological challenges.

Understanding biology by building

The ability to construct synthetic genetic circuits from the bottom up has enabled
researchers to approach questions in basic biology from a new angle. Instead of perturbing
natural pathways or model systems, biological systems can be probed by constructing
artificial circuits and examining their behavior and interactions with endogenous
machinery [70]. For example, in one investigation into why a particular circuit architecture was
selected over others that produced similar dynamics, researchers compared the native
Bacillus subtilis circuit that regulates differentiation into the competence state (when cells can take
up DNA from the environment) with an engineered circuit [71] (Figure 2a). Modeling and empirical results both indicated that, despite generating excitable
dynamics with similar shapes and frequency to those of the native competence circuit,
the engineered circuit achieved greater precision in the duration of competence. It
was further demonstrated that the greater variability in the native circuit conferred
an advantage in the form of more consistent transformation efficiency over a large
range of extracellular DNA concentrations, highlighting the effective exploitation
of noise in natural circuits [71]. Additional synthetic circuits have been constructed to study the modularity and
plasticity of MAP kinases [72,73] and the interaction between Notch and Delta critical for cell patterning during development
[74], among others [70], illustrating the utility of synthetic biology in expanding our fundamental understanding
of biology.

Figure 2.Synthetic biological circuits can aid in understanding of biology, improve biomanufacturing
productivity, and enable disease-targeted therapy. (a) The native circuit regulating competence in B. subtilis was compared with a synthetic circuit with similar dynamics to reveal architecture-specific
variability in the duration of competence and consequent differences in the consistency
of transformation efficiency over large ranges of DNA concentration [71]. (b) A synthetic protein scaffold was used to increase the biosynthesis of mevalonate from
acetyl-CoA in E. coli. The scaffold consists of three protein-protein interaction domains (GBD, the GTPase
binding domain from the actin polymerization switch N-WASP; SH3, the Src homology
3 domain from the adaptor protein CRK; and PDZ, the PSD95/DlgA/Zo-1 domain from the
adaptor protein syntrophin) in various copy numbers connected by glycine-serine linkers.
Pathway enzymes (AtoB, acetoacetyl-CoA thiolase; HMGS, hydroxymethylglutaryl-CoA synthase;
HMGR, hydroxymethylglutaryl-CoA reductase) were each fused to the ligands of one interaction
domain and recruited to the protein scaffold [39]. PTET, tetracycline-inducible promoter; PBAD, arabinose-inducible promoter. (c) A targeted therapeutic circuit was constructed by inserting an RNA aptamer near an
alternatively spliced exon harboring a stop codon in a three-exon, two-intron minigene
fused to herpes simplex virus thymidine kinase (HSV-TK). Binding of a disease marker
protein to the aptamer results in exclusion of the alternative exon, expression of
a suicide gene, and killing of diseased cells [35]. PCMV, cytomegalovirus promoter.

Expanding biomanufacturing capabilities

Biomanufacturing is one of the more compelling and immediate applications of biotechnology
that promises sustainable synthesis strategies for alternative energy sources, commodity
chemicals, and high-value specialty chemicals such as therapeutic drugs. A major challenge
of biosynthetic pathway engineering lies in balancing the levels and activities of
the many heterologous pathway enzymes to achieve optimized productivity and yield
of desired compounds in the microbial host. Synthetic biology is transforming biosynthesis
capabilities by providing new tools that support pathway construction and optimization.
For example, researchers have recently combined TAR-based assembly strategies with
sets of biosynthetic pathway parts (including enzyme coding regions, promoters, and
terminators) to demonstrate one-step, whole-pathway assembly for a variety of natural-product
pathways [75-77]. In another example, combinatorial libraries of tunable intergenic regions (TIGRs)
harboring a number of RNA regulatory elements, including terminators, RNase cleavage
sites, and stabilizing hairpins, were assembled in the non-coding regions between
three heterologous enzymes in the mevalonate biosynthetic pathway expressed from a
polycistronic transcript in E. coli. Researchers screened library variants for the TIGR sequences that resulted in optimal
relative expression levels of each enzyme to increase mevalonate production; the best
mevalonate producers decreased accumulation of a toxic intermediate and increased
growth rate [78]. Libraries of modular control elements, including promoters and RNA regulatory elements,
that have broad ranges of predictable activities have also been generated [19,79]. Recently, a library of RNase cleavage elements was used in yeast to titrate a key
enzyme and thus flux through the endogenous ergosterol pathway, which competes with
synthetic terpenoid pathways for the common precursor farnesyl pyrophosphate [80]. Finally, several new tools supporting colocalization of heterologous enzymes, such
as protein- and RNA-based scaffolds, are being used to develop pathway optimization
strategies based on spatial engineering [39,40] (Figure 2b).

Advancing next-generation therapeutics and diagnostics

By developing new strategies to interface with and manipulate natural biological systems,
synthetic biology holds exciting promise in developing new therapeutic approaches.
For instance, synthetic biologists are developing genetic circuits that link therapeutic
activities to the detection of molecular disease signals to develop targeted therapeutics
with increased efficacy and safety. In one example, a layered microRNA (miRNA)- and
transcription-factor-based logic circuit was used to distinguish a cervical cancer
cell line (HeLa) from other cell lines based on the detection of a unique miRNA profile
[57]. Positive identification of HeLa cells through this logic circuit was subsequently
linked to either expression of a reporter protein, as a model diagnostic device, or
expression of a protein that led to cell death as a model therapeutic device. In another
example, to restrict cell death to diseased cells showing hyperactive signaling, researchers
developed protein-responsive RNA devices that could detect increased signaling through
the NF-κB and Wnt pathways and transmit this information into changes in the expression
of a clinically relevant suicide gene that sensitizes cells to an apoptosis-inducing
prodrug [35] (Figure 2c). These types of autonomous sense-and-control circuits offer potential applications
in the long-term surveillance and intervention of chronic diseases, such as gout and
diabetes [68,81]. Circuits currently under development that link genetic targets to clinician-modulated
external inputs will provide an unprecedented level of temporal and spatial control
over complex therapeutic activities. For example, systems have been described that
support light-modulated glucose homeostasis [82] and drug-modulated control over in vivo gene expression [83] and T-cell proliferation [84].

Where will synthetic biology take us?

The biological parts, genetic circuits, and fabrication techniques that have been
developed and continue to be improved on offer exciting potential in diverse applications,
from environmental engineering to regenerative medicine. Synthetic biological systems
capable of detecting, reporting, and/or removing hazardous substances have been reported
[85-88], and their implementation in robust host organisms suitable for environmental release
will provide a new paradigm for environmental remediation. In the area of health and
medicine, synthetic intercellular communication systems that regulate spatial patterning,
timing of coordinated cellular responses, and tissue homeostasis have the potential
to make significant contributions to tissue engineering. Furthermore, synthetic control
circuitry may reduce the inherent tumorigenicity of stem cells [89] and improve the efficiency of induced pluripotent stem cell reprogramming [90]. Novel genetic circuits capable of guiding the ex vivo construction of complex tissues may be built in the foreseeable future as researchers
continue to unravel the systems biology behind cell-fate decisions [91,92].

Efforts in synthetic biology so far have covered a wide range of topics spanning broad
conceptual frameworks and specific circuit designs, and the future direction of synthetic
biology is by no means limited to the few areas highlighted here. However, a unifying
driving force in the field has been the desire to efficiently build biological systems,
whether to improve our fundamental understanding of biology or to provide solutions
for pressing global challenges. By developing conceptual frameworks and technical
tools for the design, construction, and characterization of novel biological systems
that can perform autonomous functions and interact with natural biological systems,
synthetic biology is poised to transform our ability to probe, understand, and manipulate
biology.

Acknowledgements

CDS is supported by funds from the National Institutes of Health, National Science
Foundation, and Defense Advanced Research Projects Agency. YYC is supported by the
Harvard University Society of Fellows.